Iron stores in steady‐state sickle cell disease children accessing care at a sickle cell disease clinic in Kumasi, Ghana: A cross‐sectional study

Abstract Background and Aims Children with sickle cell disease (SCD) have an increased risk of multiple hemotransfusions and this can predispose them to elevated iron stores. The objectives of the study were to determine the extent of elevated iron stores and the associated risk factors in a population of steady‐state SCD children in Ghana. Methods This cross‐sectional study was conducted at the pediatric sickle cell clinic at the Komfo Anokye Teaching Hospital. Complete blood count and serum ferritin assay were performed for (n = 178) steady‐state SCD children. Descriptive and multivariate logistic regression analysis were performed. Elevated iron stores were defined as serum ferritin levels >300 ng/ml. Statistical significance was considered at p < 0.05. Results The mean (standard deviation) age of the participants was 9.61 (±4.34) years, and 51% of them were males. About 17% of SCD children had elevated iron stores and receiving at least three hemotransfusions during the last 12 months was strongly associated with elevated iron stores (p < 0.001). History of chronic hemotransfusion increased the odds of having elevated iron store (adjusted odds ratio [aOR] = 11.41; 95% confidence interval [CI] = 3.11–30.85; p < 0.001) but SCD patients on hydroxyurea treatment had reduced‐odds of having elevated iron stores (aOR = 0.18; 95% CI = 0.06–0.602; p = 0.006). Moreover, red blood cell (Coef. = −0.84; 95% CI = −0.37, −1.32; p = 0.001), hemoglobin (Coef. = −0.83; 95% CI = −0.05, −1.61; p = 0.04), hematocrit (Coef. = −0.85; 95% CI = −0.08, −1.63; p = 0.03), mean cell volume (Coef. = 0.02; 95% CI = 0.01, 0.03; p = 0.001) and mean cell hemoglobin (Coef. = 0.04; 95% CI = 0.01, 0.07; p = 0.002) could significantly predict serum ferritin levels. Conclusion The magnitude of elevated iron stores was high among children with SCD in steady‐state. Red cell indices could provide invaluable information regarding the risk of elevated iron stores. SCD children who have a history of chronic hemotransfusion or had received at least three hemotransfusions in a year should be monitored for elevated iron stores.


| INTRODUCTION
Sickle cell disease (SCD) is a chronic genetic disorder that results from the inheritance of two abnormal copies of hemoglobin from carrier parents. SCD presents in several phenotypes such as the severe homozygous form (HbSS) and the less symptomatic heterozygous variants (HbSC, HbSD, HbSO-Arab, and HbS/beta-thalassemia) 1,2 These abnormal hemoglobins are due to a single amino acid substitution in the β-globin chain of normal haemoglobin (HbA) i.e., glutamic acid is replaced with valine or lysine in the 6th position of the β-globin chain, thereby producing abnormal HbS or HbC, respectively 3,4 At low oxygen tension, HbS polymerizes and converts the flexible biconcave red blood cells (RBCs) into irreversible rigid, sickle-shaped RBCs or RBCs crystalized in the case of HbC that obstructs blood flow in the microcirculation. [2][3][4] This causes clinical symptoms such as vaso-occlusive pain episodes (VOPE), severe pallor, dactylitis, acute chest syndrome (ACS), stroke, and organ injuries which often warrant hospital admissions for disease management 2,4,5 Every year, over 300,000 babies are born with SCD worldwide; and approximately 5% of the global population are healthy carriers of the gene for SCD 1,6 In Africa, over 200,000-300,000 children born every year have SCD, with 75%-80% of these children found in sub-Saharan Africa 1,6-8 Furthermore, it is reported that only about 20% of newborns with SCD survive to their second birthday in Africa 3,9 Until recently that it emerged, SCD can be cured, the disease has been managed with a medication regimen of vitamins, folic acid, penicillin V, and hydroxyurea (mostly in HbSS patients) every day 8,10 However, hemotransfusion therapy has also been accepted as the standard therapy for the management of severe cases such as anemia, ACS and stroke. [11][12][13] While this therapy effectively reduces the risk of SCD complications, elevated iron stores or iron overload is a dreaded and inevitable clinical consequence of multiple transfusions 3,14 For every unit of whole blood transfused, the body receives about 200-250 mg of iron 15,16 Besides, iron is disproportionately released into the body from chronic hemolysis and increased absorption of iron from the gastrointestinal tract 17,18 Since the body has no excretory mechanism for the excess iron 17,18 it accumulates which causes elevated iron stores or iron overload. The accumulation of excess iron in tissues or organs produces reactive oxygen species in the cytoplasm through the Fenton and Haber-Weiss reaction, which consequently lead to mitochondrial damage, disruption of the electron transport chain, peroxidation of lipids and cell membrane damage 19,20 This eventually causes apoptosis of the target organs with time, leading to complications such as endocrine, liver and heart failure, which is the commonest cause of death due to iron overload. 19,21 Generally, it is reported that patients who are hemotransfused with at least 10 units of blood are at significant risk of elevated iron stores or iron overload 16,22 Nonetheless, findings from several studies show that receiving at least three hemo transfusions per year by a sickle cell patient is significantly associated with elevated iron stores. [23][24][25] Elevated iron stores remain a burden in several parts of Africa, particularly holoendemic malaria settings. Cross-sectional studies conducted among 70 Congolese 25 and 85 Nigerian 23 children with SCD showed that nearly 21% had iron overload. In a retrospective study to analyze the clinical and/or autopsy findings of 141 adult SCD patients at postmortem in Howard University, Washington, DC, about one-third of the patients had iron overload and 7% of deaths were associated with iron overload. 26 Elsewhere in Atlanta, Georgia, a study retrospectively reported that 22 out of 387 young adults with SCD died and 10 (45%) of the deaths caused by chronic organ failure were all due to chronic iron overload (end-stage liver disease in 8 patients and congestive heart failure in 2 patients). 27 Ferritin is the principal storage form of iron in the body. Thus, serum ferritin measurement can be used as a proxy of iron status in steady-state individuals, and it is the most convenient laboratory test to estimate iron stores in resource-limited settings. According to Olufemi et al. 23 and Odunlade et al., 24 iron overload in Nigerian children with SCD is elevated beyond a ferritin level of 300 ng/ml. In Ghana, SCD remains a major public health concern where 2% (approximately 15,000) of all newborns have SCD and more than half of the patients are HbSS. 28 While there has been more research into iron deficiency in patients with SCD, the burden of elevated iron stores or iron overload in sickle cell patients has not been investigated locally.
In a holoendemic malaria setting like Ghana, children with SCD have an increased risk of multiple or chronic hemo transfusions which are mostly simple blood transfusions. Thus, these children may have an increased risk of elevated iron stores. To the best of our knowledge, this is the first time a study has investigated elevated iron stores among children with SCD in Ghana. The objectives of this study were to assess the extent of elevated iron stores and associated risk factors in a population of steady-state children with SCD accessing care in a specialized SCD management clinic in Kumasi, Ghana. Data generated from this study would help clinicians to provide timely medical diagnoses and inform the management of elevated iron stores in children with SCD in the country.

| Study design
This was a hospital-based cross-sectional study that involved only quantitative methods of data collection and laboratory analysis of blood samples. The data was collected over a period of 4 months, from August to December 2021.

| Study area
The study was conducted at the pediatric SCD clinic at the Komfo Anokye Teaching Hospital (KATH). KATH is a tertiary and major referral hospital located in the Kumasi Metropolis, the regional capital of the Ashanti region of Ghana. 29 The hospital serves many areas in the middle, northern, and southern zones of the country, and it has a bed capacity of about 1200. 29 The Pediatric SCD clinic is one of the largest SCD clinics in Ghana that provides outpatient services for children from 0 to 18 years. 29 Until the recent decentralization of SCD clinics in the country, the pediatric SCD clinic in KATH was the only clinic serving the northern, middle, and southern zones, hence it has a well-diversified population coverage.

| Study population
The population targeted for the study were children with confirmed SCD attending a clinic visit at the pediatric SCD clinic at KATH.
Children with SCD of all sexes, in a steady-state and from the ages of 3 to 18 years were recruited and included in the study. Steady-state was defined as the period when the patient with SCD is free of infection, pain, or other disease processes. 30 However, all children with SCD who experienced VOPE in the last 3 months or currently experiencing inflammation, had been hemo transfused in the last 3 months before recruitment, and refused to give assent or the caregiver refused to consent were excluded from the study.

| Sample size estimation and sampling technique
The minimum sample size estimated for the study was 177. This was determined using the Fisher's formula for sample size calculation, N = [z 2 p (1−p)]/d 2 , 31 and based on the assumed prevalence of iron overload i.e. 21%, reported by Olufemi et al. 23 in steady-state children with SCD. Specifically, N was the minimum sample size estimated; z was the point of the standard normal distribution curve which was set at 1.96 (95% confidence interval [CI]); p was the assumed prevalence rate; and d was the degree of precision which was set at 6%. After review of medical records and complete blood count, 178 participants met the eligibility criteria and were included in the analysis.
Systematic random sampling technique was employed to recruit the study participants. SCD clinic registers were reviewed for the total number of patients who visited the clinic within the period of data collection. Using this number, the sampling frame (nth) was calculated from the quotient of the number of patients who visited the clinic (N) and the estimated sample size (n) i.e., nth = N/n. 32 Therefore, participants were chosen at random after every "nth" count of patients visiting the clinic and in the subsequent days of the recruitment period.

| Study procedure
The selected SCD patients were screened for eligibility. Permission was obtained from caregivers whose child met the inclusion criteria, and caregivers or patients more than 7 years were consented or assented respectively. Five milliliters of blood samples were collected from each patient, i.e., 2 and 3 ml were transferred into an Erythrocyte sedimentation rate (ESR) was also performed using the fresh EDTA blood samples to screen for possible inflammation. The fresh EDTA blood samples Following centrifugation of the sample in the SS tube, the serum was separated and stored in a minus 80°C freezer until a pool sample of 178 was obtained for the ferritin assay.

| Data collection
Caregivers and/or study participants were interviewed with an electronic semi-structured questionnaire hosted on the School of Medicine and Dentistry, KNUST Research Electronic Data Capture (REDCap) server. 33 REDCap is a secure web application for building and managing online surveys and databases. To ensure that quality data was collected, the questionnaire was answered by caregivers on behalf of study participants below the age of 13 years, while participants from 13 years and above responded to the questionnaire and in some cases were assisted by their caregivers. Moreover, questions were interpreted in the local language where necessary for easy comprehension. Medical records were reviewed for each study participant for the clinic visit to confirm their steady state and AMANOR ET AL. | 3 of 12 complete other relevant variables. The questionnaire was categorized into background characteristics, clinical characteristics, and current treatment. Furthermore, the laboratory results of study participants were entered into a Laboratory Documentation Sheet which was also hosted on REDCap so that each study participant has a point source document. The questionnaire was pilot tested at the pediatric SCD clinic in the Maternal and Child Health Hospital. A total of 10% of the calculated sample size was used for the pilot test to accomplish a reasonable power to ensure the reliability and validity of the questionnaire.

| Dependent/outcome variables
The outcome variable "elevated iron stores" was measured binary using serum ferritin ≤300 ng/ml and >300 ng/ml responses. Elevated iron stores were defined as serum ferritin >300 ng/ml according to previous studies 23,24,34 Moreover, log transformation of serum ferritin concentration was carried out and used as the outcome variable in a linear regression analysis.

| Independent/predictor variables
The predictor variables included demographic and clinical characteristics of study participants. Demographic characteristics such as age and gender were considered relevant in this study. The age of the study participants was categorized according to the WHO AGE categorization, i.e., <5 years, 5-9 years, 10-14 years, and ≥15 years. 35 The clinical characteristics such as SCD genotype, VOPE in the last 12 months, frequency of hospitalization in the last 12 months, frequency of hemotransfusion in the last 12 months, ever been hemotransfused, history of chronic transfusion and hydroxyurea treatment were considered relevant for this study based on literature.
Chronic transfusion was defined as receiving blood transfusions on a regular basis, i.e., at least one hemotransfusion in every month. 11 Moreover, RBC, hemoglobin, hematocrit, mean cell volume, and mean cell hemoglobin were considered for the linear regression.

| Statistical analysis
The data were analyzed using Stata (STATA/SE version 16.0).
Descriptive statistics were performed for participant demographics, clinical, and laboratory characteristics. Categorical variables were expressed as frequency/percentage while continuous variables were expressed as mean/standard deviation and median/interquartile range (IQR) according to whether the distribution of the variables was Gaussian or not Gaussian, respectively. The Mann-Whitney test was computed to compare gender differences with regards to serum ferritin levels. The Kruskal-Wallis test was performed to compare age differences and SCD genotype with regards to serum ferritin levels.
The relationship between serum ferritin levels and frequency of hemotransfusions per year was established by Kendall's correlation and graphically presented by a boxplot. The correlation between CBC indices and serum ferritin levels was established by Kendall's correlation. Moreover, a linear regression was used to predict the relationship between CBC indices and serum ferritin levels and graphically presented by a scatterplot. Bivariate and multivariate logistic regression analysis were performed to calculate the crude odds ratio and adjusted odds ratio at 95% CI between the dependent variable and all predictor variables. Statistical significance was considered at p < 0.05.

| Ethical approval
Ethical clearance was obtained from the KATH Institutional Review Board (IRB) with reference: KATH IRB/AP/061/21. The background, aims, and study procedures were thoroughly explained to caregivers and patients where necessary in a language they can comprehend.
Written informed consent and assent were obtained before any study participant was enrolled into the study.  Kruskal-Wallis test. This was followed by the Conover-Iman test and the pairwise comparison showed the main difference was between the age groups of 10-14 years and 5-9 years (p = 0.009) as summarized in Table 3.   This is presented in Figure 2.

| Factors associated with elevated iron stores among study participants
To establish the risk factors associated with elevated iron stores among the study participants, a crude analysis was performed. Here, the data revealed that the frequency of hospitalization in the last 12 months, ever been hemo-transfused, chronic hemotransfusion and frequency of hemotransfusion in the last 12 months were significant factors associated with elevated iron stores in steady-state SCD of the participants. The pairwise comparison showed that the median difference was dominantly observed between participants from the ages of 10-14 years and 5-9 years. Unlike our findings, Makulo et al. 25 found no significant difference in serum ferritin levels between the various age categories of children with SCD and this may be due to the smaller sample size of their study (N = 70). Our findings confirm that serum ferritin level in children with SCD is a function of age.
Hematological parameters are crucial in disease diagnosis or prognosis because changes in these parameters above or below normal range may account for clinical complications in patients. In SCD patients, hematological parameters are evaluated regularly on clinic visits to ensure good management of the disease. Thus, we believe it is imperative to examine the association between serum ferritin and hematological parameters so that it can inform physicians when making decisions on iron store diagnosis. The study found no correlation between serum ferritin and differential white blood cell counts. These findings were consistent with the reports of several studies that were conducted on children with SCD and other chronic non-communicable diseases 25,36,37 Ferritin is an acute-phase protein that is elevated in the presence of infection or inflammation.
Differential white blood cells are major components of the body's defensive system that may become abnormal in the presence of an beyond 500 ng/ml or 1000 ng/ml 25,46 Furthermore, three or more hemotransfusions and being hospitalized for at least four times in the last 12 months were likely to cause elevated iron stores in SCD children in the present study. These findings were similar to the reports of other studies 25,47 We anticipated these observations because the more an SCD patient is hospitalized, the likelihood the patient will be hemotransfused, given that SCD patients have an increased risk of hemotransfusion.
Moreover, the present study showed that ever been hemotransfused and a history of chronic hemotransfusion was significantly associated with elevated iron stores and this is consistent with several studies 45,47,48 When adjusted for all covariates, children with SCD who had ever been hemotransfused and chronically transfused in the past were about 9 and 11 times likely to have elevated iron stores, respectively. Eveline et al. showed that iron overload is more concerned with the male gender and this is so because blood loss during menstruation results in significant iron loss in females, unlike their male counterparts who have no robust mechanism to remove iron from the body. 49 However, this was not the scenario in our study as we observed males have reduced odds of developing elevated iron stores. This is interesting and warrants further investigation to unravel the underlying mechanisms. On the other hand, SCD children who were on hydroxyurea treatment had reduced odds of developing elevated iron stores. These findings were similar to the report by Italia et al. 50 which showed that serum ferritin decreased significantly in SCD patients after 2 years of hydroxyurea therapy and also in mice models treated with hydroxyurea. 51 Makulo et al. 25 found no significant association between elevated iron stores and hydroxyurea treatment in SCD children. The differences relative to the results of other studies could be attributed to compliance with hydroxyurea treatment. It is reported that hydroxyurea is a radical scavenger that has hydroxamate function properties and hence can act as an iron chelator. This may explain the observation in the present study; however, we strongly suggest further studies to provide more insight into the iron chelation properties of hydroxyurea.

| LIMITATIONS
This study provided valuable information that may be used by local physicians to monitor elevated iron stores or iron overload in children with SCD. However, the study has some limitations that can strengthen our findings when they are addressed in future studies.
First, we did not perform liver biopsy or magnetic resonance interference which has been shown to provide an excellent assessment of elevated iron stores (iron overload) 43,45 to confirm our findings. Second, serum creatinine reactive protein levels were not measured to confirm the steady state of the study participants due to limited funds. However, this was complemented by reviewing the medical records of the participants clinic visit and performing ESR test to further screen those who were recruited. Third, the findings could have been strengthened by providing information on the products of blood transfused (whole blood, pack-cell, and plasma) and the exact volume of blood received by patients. However, some of these factors were not available in the medical records of some study participants and some were also not hemotransfused at the facility, hence these factors were not available for them.

| CONCLUSION
The magnitude of elevated iron stores is high among children with SCD in the present study and this must be given the needed attention. SCD children who have a history of chronic hemotransfusion or had received at least three hemotransfusions in a year should be monitored for elevated iron stores. Red cell indices can provide invaluable information regarding the risk of iron store elevation and can serve as a prompt for physicians to monitor the elevation of iron stores in SCD children. We suggest further studies that take into consideration the limitations of this present study and a detailed study on the iron chelation effect of hydroxyurea on iron stores in SCD patients.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data and materials are available in the corresponding author's institution and will be made available upon formal request.

TRANSPARENCY STATEMENT
The lead author Ernest Amanor affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.